Angiogenesis, the sprouting of new capillary blood vessels from existing vasculature, is a complex biological process of critical importance to the treatment of numerous pathologies and the success of tissue engineering. Cell-based strategies to promote therapeutic vascularization have shown pre-clinical and clinical success, particularly for the treatment of critical limb ischemia, and typically involve the delivery of a single mesenchymal cell type to ischemic sites to stimulate angiogenesis. By contrast, strategies to vascularize engineered tissues typically involve two cell types, with endothelial cells (or their progenitors) combined with some sort of supporting stromal cell type and delivered via a hydrogel-based extracellular matrix (ECM). However, the choices of stromal cells and ECM have varied widely across studies. In the prior funding period, we used a combination of 3D cell culture models in vitro and subcutaneous implants in vivo to discover that the formation of nascent vasculature is regulated by both the biophysical properties of the ECM and the identity of the supporting stromal cells. Our data suggest that stromal cells of different origins differentially control ECM proteolysis during angiogenic sprouting, and that controlling the rate o ECM breakdown is critical to yield stable, functional vessels. In this competing renewal application, we propose to mechanistically investigate how these two critical instructive elements of the local microenvironment (i.e., the stromal cells and the ECM) influence the quantity, functional quality, and stability of new vasculature. Aim 1 will quantify the impact of stromal cell identity on the rates of ECM proteolysis and capillary morphogenesis in a fibrin-based 3D co-culture model, using active microrheology to monitor local ECM mechanics as a function of time. Aim 2 will use an engineered biomaterial platform with tunable degradative properties to determine the impact of ECM proteolytic susceptibility on the quantity and functional qualities of capillary networks. Aim 3 will investigate how stromal cell identity and ECM proteolytic susceptibility affect the quantity and quality of neovasculature in a clinically relevant ischemic model. Successful completion of these studies will enhance current understanding of the role of the microenvironment in capillary morphogenesis, and ultimately guide the judicious selection of the ideal combination of cells and ECM for the treatment of ischemic conditions.